Project Summary Pro-atherogenic stimuli such as inflammatory cytokines and disturbed flow (d-flow) significantly contribute to endothelial cell (EC) inflammation, and subsequent atherosclerotic plaque formation. Membrane-associated guanylate kinase-1 (MAGI1) is a scaffold protein that contains six PSD95/DiscLarge/ZO-1 (PDZ) domains, a guanylate kinase domain, and two WW domains flanked by the first and second PDZ domains. MAGI1 associates with the tight and adherens junction, but its cytosolic and nuclear localization has also been reported. Recently, a genome-wide association study has revealed a strong association of mutations in Magi1 gene locus with inflammatory bowel disease and psoriatic arthritis. The significant contribution of EC inflammation in regulating the phenotype and severity of these diseases, which are also clinically associated with accelerated atherosclerosis (AS) and increased risk of cardiovascular diseases, has been well established. In the preliminary data, we have found the critical role of MAGI1 post-translational modifications (PTMs) in regulating EC inflammation and apoptosis. MAGI1 S741 phosphorylarion results in the activation of Rap1 and large tumor suppressor 1 (LATS1) (but not Yes-associated protein [YAP] phosphorylation, which is a canonical pathway regulated by LATS1). MAGI1 de-SUMOylation leads to the co-translocation of MAGI1 and p90RSK to the nucleus, where they up-regulate inflammatory gene expression. In the proposed study, we hypothesize that dynamic modulation of MAGI1 PTMs (phosphorylation and de-SUMOylation) leads to cytosolic Rap1 and non- canonical LATSs signaling activation, and p90RSK nuclear translocation. MAGI1 in both cytosolic and nuclear compartments plays critical roles in regulating EC inflammation and apoptosis and subsequent atherogenesis. Aim 1 will identify the mechanism by which MAGI1 S741 phosphorylation promotes EC inflammation and apoptosis. Aim 2 will determine the role of MAGI1 de-SUMOylation in nuclear translocation and promoting EC inflammation. In aim 3, we will determine the role of MAGI1 and LATS1/2 in accelerating AS. The concept of the MAGI1 PTMs including phosphorylation- and de-SUMOylation-induced EC inflammation is novel and highlights the importance of determining how this PDZ domain-containing molecule and p90RSK coordinately regulate EC inflammation and apoptosis. The long?term goals of this project are to identify the role of MAGI1 phosphorylation and (de)SUMOylation in regulating MAGI1 subcellular localization and MAGI1-dependent signaling and to elucidate the molecular mechanisms by which MAGI1 induces EC inflammation and apoptosis and promotes atherogenesis. This will provide deeper understanding of the basic signaling responsible for the poor outcome of AS-related cardiovascular diseases.